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Heteromultimers with reduced or silenced effector function

a heterodimer and effector function technology, applied in the field of therapeutic antibody design, to achieve the effect of reducing the effector function of an antibody construct and reducing binding

Active Publication Date: 2016-04-14
ZYMEWORKS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a new type of antibody that has reduced binding to certain proteins in the body. This is achieved by modifying the lower hinge region of the antibody's Fc polypeptides. The modifications can increase or decrease the net charge of the Fc polypeptides, which reduces their binding to Fc receptors and C1q protein. This new antibody design has potential use in reducing the effector function of antibodies, making them safer and more effective for therapeutic purposes.

Problems solved by technology

For certain disease indications, however, effector functions mediated by the Fc region of the antibody can cause undesirable adverse effects and thus efforts have been made to engineer antibodies with reduced or silenced effector functions.

Method used

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  • Heteromultimers with reduced or silenced effector function
  • Heteromultimers with reduced or silenced effector function
  • Heteromultimers with reduced or silenced effector function

Examples

Experimental program
Comparison scheme
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example 1

Preparation and Expression of Antibody Constructs (Heteromultimers)

[0234]The following antibody constructs were prepared. All antibody constructs were based on the sequence of the wild-type anti-Her2 antibody trastuzumab (see FIG. 5, SEQ ID NO:2 for wild-type trastuzumab Heavy chain amino acid sequence, SEQ ID NO:3 for wild-type trastuzumab light chain sequence) with the following added modifications in the heavy chain CH3 domain introduced in order to promote the formation of a heterodimer Fc domain with increased stability as compared to a CH3 domain that does not comprise amino acid mutations.

Chain A: T350V / L351Y / S400E / F405A / Y407V, and

Chain B: T350V / T366L / N390R / K392M / T394W

[0235]This construct, with the above modifications is referred to as v791. All sequences described herein are numbered using the EU numbering system.

[0236]Additional variants were constructed based on v791, with amino acid modifications in the hinge region and or CH2 domain of the heavy chain as shown in Table A...

example 2

Asymmetric Antibody Constructs Based on Trastuzumab do not Bind to FcγR

[0240]The ability of the asymmetric antibody constructs to bind to FcγRIIaH, FcγRIIaR, FcγRIIb FcγRIIIaF, FcγRIIIaV, and FcγRIa was assessed by surface plasmon resonance (SPR).

[0241]Affinity of FcγRs to antibody Fc was measured by SPR using a PrateOn XPR36 at 25° C. with 10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, and 0.05% Tween 20 at pH 7.4. Recombinant HER-2 was captured on the activated GLM sensorchip by injecting 4.0 μg / mL in 10 mM NaOAc (pH 4.5) at 25 μL / min until approx. 3000 resonance units (RUs) were immobilized with the remaining active groups quenched. 40 μg / mL of purified HER-2 / neu-based antibodies were indirectly captured when injected at 25 μL / min for 240 s (resulting in approx. 500RUs) following a buffer injection to establish a stable baseline. FcγRs were injected at 60 μL / min for 120 s with a 180 s dissociation phase to obtain a set of binding sensograms. Resultant kD values were determined from bindi...

example 3

Asymmetric Antibody Constructs Based on Trastuzumab do not Bind to C1q

[0244]The ability of the asymmetric antibody constructs to bind to C1q was tested as follows. Human C1q was purchased from GenWay Biotech (San Diego, Calif.). SPR chip immobilization of antibodies was as described in Example 2. 30 nM C1q injected over mAb variants captured onto a HER2 SPR surface using standard protocols as also described in Example 2. The results are shown in Table C below.

TABLE CResults of C1q binding assayVariantC1q1WTyesControl / 1051NBAAC1partialAAC2NBAAC3NBAAC4NBAAC5NBAAC6NBAAC7NBAAC8NB1C1q is a hexamer of heterotrimers witha potential stoichiomentry mAb:C1q of 6:1. The binding kinetics were very complex, and a proper Kd could not be determined. Receptor was tested at 30 nM. ‘partial’ means diminished binding, ‘NB’ means no detectable binding

[0245]All of the variants showed undetectable binding to C1q, except for AAC1 which showed decreased, but detectable binding to C1q.

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Abstract

Provided herein are heteromultimer constructs with reduced or silenced effector function. In an embodiment is provided a heteromultimer construct comprising an IgG Fc construct having a first and a second Fc polypeptide, each Fc polypeptide comprising a modified lower hinge region wherein: the modified lower hinge region of said first Fc polypeptide comprises at least one amino acid modification, the modified lower hinge region of said second Fc polypeptide comprises at least one amino acid modification which is different from at least one amino acid modification of said first Fc polypeptide, and the IgG Fc construct displays reduced binding to all Fcγ receptors and to C1q protein as compared to a corresponding parent IgG Fc construct. Also provided are methods of producing such heteromultimer constructs, and methods of reducing ADCC for an antibody construct by reducing effector function.

Description

CROSS-REFERENCE[0001]This application claims the benefit of U.S. Provisional Application No. 61 / 829,973, filed May 31, 2013, which application is incorporated herein by reference in its entirety for all purposes.INTRODUCTION[0002]2.1. Field of the Invention[0003]The present invention relates to the field of therapeutic antibody design and specifically to polypeptides comprising a heterodimeric Fc region which has been modified in order to silence effector functions mediated by the Fc region.[0004]2.2 Background of the Invention[0005]Therapeutic antibodies have been developed for the treatment of many disease indications. In some of these cases the efficacy of the therapeutic antibody results, at least in part, from the ability of the Fc region of the antibody to mediate one or more effector functions. These effector functions result from the interaction of antibodies and antibody-antigen complexes with cells of the immune system to stimulate a variety of responses, including antibod...

Claims

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Application Information

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IPC IPC(8): C07K16/00C07K16/28C07K16/32
CPCC07K16/00C07K16/32C07K16/2887C07K2317/53C07K2317/94C07K2317/92C07K2317/41C07K2317/526C07K2317/71A61P17/00A61P17/02A61P17/06A61P19/02A61P25/00A61P25/04A61P25/28A61P29/00A61P3/00A61P37/00A61P7/04A61P9/00A61P9/04A61P9/10C07K16/468
Inventor ESCOBAR-CABRERA, ERIC
Owner ZYMEWORKS INC
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